A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences (‘target sequences’) in the presence of a large excess of non-variant sequences (‘reference sequences’). As an example, sensitive and confident detection of trace disease-associated mutant alleles among an excess of normal alleles has become essential for early or confirmatory diagnoses, therapeutic decisions, disease monitoring, and prognostic stratification. However balancing high sensitivity with reliability and practicality of use remains challenging.
Sanger sequencing, the robust standard method, can only detect approximately 10-20% of mutant alleles in a background of normal alleles, whereas other, more sensitive, PCR-based assays such as restriction fragment length polymorphism, amplification refractory mutation system PCR (ARMS-PCR), denaturing high-performance liquid chromatography, and real-time PCR could be more limited. Amplification refractory mutation system PCR, the most widely used method, is an amplification strategy in which a PCR primer is designed to discriminate among templates that differ by a single nucleotide residue. This method is simple and time efficient but is sequence-specific, requiring to know the mutation to detect beforehand, and sometimes produces serious false-positive results.
There have been efforts to develop new molecular technologies aimed at overcoming the inherent drawbacks of prior methods. There has been particular interest in the innovation of PCR stages that enable nondestructive selection and enrichment of mutant alleles, as this can improve sensitivity and credibility of downstream assays, such as standard sequencing analysis. These recent enrichment PCR techniques include PCR clamping mediated by peptide nucleic acid (PNA) (Sun et al, 2002) or locked nucleic acid (LNA) (Dominguez et al, 2005), and co-amplification at lower denaturation temperature PCR (Li et al, 2008). Each technique has its own strengths and limitations in regard to the cost, availability, or enrichment efficiency.
In Lee et al, 2011, the authors describe a simple and practical enrichment technique, called mutant enrichment with 3′-modified oligonucleotides (MEMO). The concept of this technique is similar to that of PNA/LNA-mediated PCR clamping, but the PNA or LNA is replaced by 3′-modified oligonucleotides that are much less expensive and are easy to design. Briefly, two generic primers and one blocking primer constitute the PCR reaction mixture. On the 3′-end of the blocking primer, an extension-inhibiting compound such as a C3 spacer, a C6 amine, or a phosphate is attached so that PCR cannot extend the DNA via the blocking primer. The blocking primer encompasses the target mutation site and is complementary to the wild-type sequence. One of the two generic primers overlaps with the blocking primer by several bases, neighboring the target mutation site, and thus is in competition with the blocking primer. The DNA binding of the blocking primer, which is designed to have a higher melting temperature and to be used in a higher concentration in the reaction mixture than the generic primer with which it competes, dominates for wild-type sequence, whereas its affinity for mutant sequences is markedly reduced due to mismatches. The loss of competition of the blocking primer enables selective amplification of mutant sequences by the generic primer pair.
This MEMO-PCR was employed to identify a rare 3 bp BRAF gene deletion in a thyroid nodule (Jang et al, 2012).
However there remained a need for a method of detection with improved specificity, in particular when the variant target sequences carry nucleotide substitution(s). Indeed such sequences had been poorly discriminated so far.